1. Field of the Invention
The present application relates to modulating the activity of the innate immune system. More particularly the application relates to modulating innate immune system receptor signaling using microRNA and by inhibiting microRNA activity.
2. Description of the Related Art
Inflammation is a complex, highly regulated defense reaction orchestrated in response to invading pathogen or injury. Inflammation usually proceeds in several sequential stages: it starts with localization of infectious agent, a step that is aimed at preventing of the spread of pathogen to other tissues and organs, and is followed by recognition of the ‘danger’ signal and activation of the innate immune system and recruitment of specialized immune cells to the site of infection; it ends with the elimination of the pathogen and infected cells by the immune cells of the host, termination of the immune response and repair of damaged tissue. Initiation, smooth transition from one stage to another and especially termination of the inflammatory process is fully dependent on coordinated activities of multiple cell types at the site of inflammation and within the immune system. Ultimately, this coordination is achieved through the ability of cells to communicate with each other through signals in the form of tissue mediators and cytokines.
The initial step of detection of pathogenic organisms invading a host is mediated by the innate immune system. Unlike adaptive immunity, innate immunity does not recognize every possible antigen. Instead, it is designed to recognize a few highly conserved structures present in many different microorganisms. The structures recognized are called pathogen-associated molecular patterns and include, for example, LPS from the gram-negative cell wall, peptidoglycan, lipotechoic acids from the gram-positive cell wall, the sugar mannose (common in microbial glycolipids and glycoproteins but rare in those of humans), bacterial DNA, N-formylmethionine found in bacterial proteins, double-stranded RNA from viruses, and glucans from fungal cell walls. These microbial molecules are sensed by pattern recognition receptors of the Toll/Toll-like receptor (TLR) family, which activate the innate immune response. The binding of a microbial molecule to its TLR transmits a signal to the cell's nucleus inducing the expression of genes coding for the synthesis of intracellular regulatory molecules called cytokines. The cytokines, in turn, bind to cytokine receptors on other defense cells, thus further shaping and enhancing the inflammatory reaction.
Upon binding of their cognate ligands, TLRs recruit adaptor molecules to their intracellular signaling domain, leading to the activation of numerous kinases, activation of several transcriptional factors (e.g. AP-1, NF-kB and IRF3/7), and direct regulation of immune-responsive genes. The TLR signaling cascade starts when an adaptor protein MyD88 is recruited to the receptor complex, followed by its association with the IL-1R-associated kinase 1 (IRAK1). Activated 1RAK1 binds TNF receptor-associated factor (TRAF6), thereby triggering the activation of the downstream effector molecules in the AP-1 and NF-kB activation pathways. NF-kB is a key transcriptional factor that regulates all aspects of the innate immune response from synthesis of pro-inflammatory cytokines such as IL-β and TNFα to regulation of immune cell migration to remodeling of the tissues after the successful termination of inflammatory response.
Activation of TLR downstream targets like cytokines TNF and IL-1 can result in a systemic disorder like sepsis or local, chronic inflammation disease like rheumatoid arthritis or inflammatory bowel syndrome. One of the classical modes of regulation of signaling in nature is a transcriptional feedback loop, a mechanism where activation of a certain transcriptional factor leads to a transcriptional activation of a gene that modulates signaling towards activation of this same transcriptional factor. TLRs activate hundreds of genes at the transcriptional level, some of which are secreted molecules that serve as means of communication with other cells, while others are involved in modulation of TLR receptor signaling or mediate crosstalk between TLR receptors and other signaling systems.
MicroRNAs (miRNAs) are a recently discovered class of small RNA molecules that are emerging as potent regulators of multiple aspects of cellular function. MicroRNAs (miRNAs) are evolutionally conserved class of endogenous 22-nucleotide RNAs involved in post-transcriptional gene repression. Bartel, D. P., Cell 116, 281-97 (2004); Ambros, V., Nature 431, 350-5 (2004); Farh, K. K. et al., Science 310, 1817-21 (2005). In animals, miRNAs are processed from larger primary transcripts (pri-miRNA or pri-miR) through an approximate 60-bp hairpin precursor (pre-miRNA or pre-miR) into the mature forms (miRNA) by two RNAse III enzymes, Drosha and Dicer. Gregory, R. I. et al., Nature 432, 235-40 (2004); Chendrimada, T. P. et al., Nature 436, 740-4 (2005). The mature miRNA is loaded into the ribonucleoprotein complex (RISC), where it typically guides the downregulation of target mRNA through base pair interactions. Pri-miRNAs are transcribed by RNA polymerase II and predicted to be regulated by transcription factors in an inducible manner. Lee, Y. et al., Embo J 23, 4051-60 (2004); Fazi, F. et al., Cell 123, 819-31 (2005); O'Donnell, K. A., et al., Nature 435, 839-43 (2005). While some miRNAs show ubiquitous expression, others exhibit only limited developmental stage-, tissue- or cell type-specific patterns of expression. Pasquinelli et al., Curr Opin Genet Dev 15, 200-5 (2005). In mammals, miRNAs have been associated with diverse biological processes, such as cell differentiation (Chen, et al., Science 303, 83-6 (2004); Monticelli, S. et al. Genome Biol 6, R71 (2005); Esau, C. et al., J Biol Chem 279, 52361-5 (2004)), cancer (Calin, G. A. et al., Proc Natl Acad Sci USA 101, 2999-3004 (2004); Lu, J. et al., Nature 435, 834-8 (2005); He, L. et al., Nature 435, 828-33 (2005)), regulation of insulin secretion (Poy, M. N. et al., Nature 432, 226-30 (2004)), and viral infection (Lecellier, C. H. et al., Science 308, 557-60 (2005); Sullivan, C. S. and Ganem, D. Mol Cell 20, 3-7 (2005)). Studies in plants have shown that miRNAs can be involved in the responses to a variety of environmental stresses.
The human genome contains two miRNA-146 genes with high sequence of homology, miR-146a (Cai, X. et al., Proc Natl Acad Sci USA 102, 5570-5 (2005), which is herein expressly incorporated by reference) and miR-146b (Bentwich, I. et al. Nat Genet 37, 766-70 (2005), which is herein expressly incorporated by reference). The mature forms of these genes differ only by two nucleotides.